Disruption of HNF1α binding site causes inherited severe unconjugated hyperbilirubinemia.

Crigler-Najjar syndrome presents as severe unconjugated hyperbilirubinemia and is characteristically caused by a mutation in the UGT1A1 gene, encoding the enzyme responsible for bilirubin glucuronidation. Here we present a patient with Crigler-Najjar syndrome with a completely normal UGT1A1 coding region. Instead, a homozygous 3 nucleotide insertion in the UGT1A1 promoter was identified that interrupts the HNF1α binding site. This mutation results in almost complete abolishment of UGT1A1 promoter activity and prevents the induction of UGT1A1 expression by the liver nuclear receptors CAR and PXR, explaining the lack of a phenobarbital response in this patient. Although animal studies have revealed the importance of HNF1α for normal liver function, this case provides the first clinical proof that mutations in its binding site indeed result in severe liver pathology stressing the importance of promoter sequence analysis.

Spectrum of UGT1A1 mutations in Crigler-Najjar (CN) syndrome patients: identification of twelve novel alleles and genotype-phenotype correlation.

Crigler-Najjar syndrome types I and II (CN1 and CN2) are usually inherited as autosomal recessive conditions and are characterized by non-hemolytic unconjugated hyperbilirubinaemia. CN1 is the most severe form, associated with the absence of hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1) activity. CN2 presents intermediate levels of hyperbilirubinaemia as a result of an incomplete deficiency of hepatic UGT1A1 activity. Here, we present the analysis of UGT1A1 gene in 31 unrelated Crigler-Najjar (CN) syndrome patients. This analysis allowed us to identify 22 mutations, 12 of which were not previously described, expanding the spectrum of known UGT1 mutations to 77. Novel mutations, considered pathogenic, including one nonsense mutation, two altered splice sites, one single base deletion and nine missense mutations were identified in coding exons of the UGT1A1gene and flanking introns. Several novel missense mutations localize in critical domain of UGT1A1 enzyme. In addition, the evaluation of Gilbert-type promoter of UGT1A1in Crigler-Najjar (CN) syndrome patients was performed. The polymorphisms of the promoter region can modify the UGT1A1 mutation phenotype. This study represents the molecular characterization of the largest cohort of Italian Crigler-Najjar Gilbert syndrome patients studied so far; increase the mutational spectrum of UGT1A1 allelic variants worldwide and provide a new insight useful for clinical diagnosis and genetic counseling.

Coding defect and a TATA box mutation at the bilirubin UDP-glucuronosyltransferase gene cause Crigler-Najjar type I disease.

Mutations at the bilirubin UDP-glucuronosyltransferase (transferase) gene in a severely hyperbilirubinemic Crigler-Najjar (CN) type I individual was compared with that in a moderately hyperbilirubinemic CN II individual. The CN-I (CF) patient in this study sustained a TATA box insertional mutation which was paired with a coding defect at the second allele, unlike all coding defects previously seen in CN-I patients. The sequence of the mutant TATA box, [A(TA)8A], also seen in the CN-II patient, was compared with that at the wild-type box, [A(TA)7A]. Transcriptional activity with [A(TA)8A] was 10-15% that with the wild-type box when present in the -1.7 kb upstream regulatory region (URR) of the bilirubin transferase UGT1A1 gene which was fused to the chloramphenicol acetyl transferase reporter gene, pCAT 1.7H, and transfected into HepG2 cells. Also, a construct with a TA deletion, [A(TA)6A], was prepared and used as a control; transcriptional activity was 65% normal. The coding region defect, R336W, seen in CF (CN-I) was placed in the bilirubin transferase UGT1A1 [HUG-Br1] cDNA, and its corresponding protein was designated UGT1A1*32. The UGT1A1*32 protein supported 0-10% normal bilirubin glucuronidation when expressed in COS-1 cells. The I294T coding defect seen at the second allele in SM (CN-II) generated the UGT1A1*33 mutant protein which supported 40-55% normal activity with a normal Km (2.5 microM) for bilirubin. The hyperbilirubinemia seen in SM decreased in response to phenobarbital treatment, unlike that seen in CF. Parents of the patients were carriers of the respective mutations uncovered in the offspring. The TATA box mutation paired with a deleterious missense mutation is, therefore, completely repressive in the CN-I patient, and is responsible for a lethal genotype/phenotype; but when homozygous, i.e. paired with itself, as previously reported in the literature, it is far less repressive and generates the mild Gilbert's phenotype.

Analysis of bilirubin uridine 5'-diphosphate (UDP)-glucuronosyltransferase gene mutations in seven patients with Crigler-Najjar syndrome type II.

Crigler-Najjar syndrome (CN) type II is caused by a reduction in hepatic bilirubin uridine 5'-diphosphate (UDP)-glucuronosyltransferase activity. Recently, there has been progress in mutation analysis of patients with CN type II. Here, we analyzed both the coding and the promoter regions of the gene in seven Japanese patients with CN type II from five unrelated families. The mutations found in this study were classified into three types. The first type was composed of double homozygous missense mutations (Gly71Arg and Tyr486Asp) in exons 1 and 5. These mutations, which were detected in five patients from three unrelated families, were the commonest. The second type, which was detected in one patient, consisted of a single homozygous missense mutation (Arg209Trp) in exon 1. The third type, which was detected in one patient and was a new type of mutation combination, was composed of a homozygous insertion mutation of the TATAA element and a heterozygous missense mutation (Pro229Gln) in exon 1. Although the first and the second type of mutations are recessive, the third type appears to be dominant with incomplete penetrance, since the allele frequency of the insertion mutation of the TATAA element is very high (40%).

Contribution of two missense mutations (G71R and Y486D) of the bilirubin UDP glycosyltransferase (UGT1A1) gene to phenotypes of Gilbert's syndrome and Crigler-Najjar syndrome type II.

In our mutation analyses of bilirubin UDP glycosyltransferase (UGT1A1) gene, we encountered six patients with Crigler-Najjar syndrome type II who were double homozygotes for G71R and Y486D, a patient with Gilbert's syndrome who was a single homozygote for G71R and six patients with Gilbert's syndrome who were single heterozygote for G71R. To clarify the role of each mutation in the occurrence of the two syndromes, we made four mutant expression models. Relative UGT1A1 activity of a single homozygous model of G71R was 32.2+/-1.6% of normal, that of a single homozygous model of Y486D was 7.6+/-0.5%, that of a double homozygous model of G71R and Y486D was 6.2+/-1.6% and that of a heterozygous model of G71R was 60.2+/-3.5%. The decreased activities of the single homozygous model of G71R and the double homozygous model were at an appropriate level to be diagnosed as Gilbert's syndrome and CN-II, respectively. The activity of a single heterozygous model of G71R was somewhat high to develop to the phenotype of Gilbert's syndrome, suggesting the presence of additional factors for the etiology of Gilbert's syndrome.

Liver bilirubin UDP-glucuronosyltransferase activity in chronic nonhemolytic unconjugated hyperbilirubinemia of adults.

Ten adult patients with chronic nonhemolytic unconjugated (indirect) hyperbilirubinemia were analyzed by determining bilirubin uridine diphosphate-glucuronosyltransferase activity according to a more physiological and sensitive method (9 control cases, 0.457 +/- 0.163 nmole/mg protein/min). There was no overlap of the enzyme activities of 2 cases with Crigler-Najjar syndrome (type II) (0.006 nmole/mg protein/min on average) and 6 cases with Gilbert's syndrome (0.051 +/- 0.016 nmole/mg protein/min). The enzyme activities in 2 patients with post-hepatitic hyperbilirubinemia were within the normal range. A new classification of nonhemolytic unconjugated hyperbilirubinemia in adults is proposed according to the results of this enzyme activity and the recent data on the gene mutation of this enzyme.

[Anesthetic and postoperative care of a patient with Crigler-Najjar syndrome type II].

Crigler-Najjar syndrome is a rare inherited deficiency of bilirubin uridine diphosphate glucuronyl transferase, characterised by lifelong unconjugated hyperbilirubinemia. We report anesthetic and postoperative management of a patient with this syndrome. A 64-yr-old man with Crigler-Najjar syndrome type II underwent cholecystectomy. Preoperative laboratory values included total bilirubin 8.4 mg.dl-1 and direct-bilirubin 1.8 mg.dl-1. He received epidural anesthesia combined with general anesthesia with nitorous oxide and isoflurane. He had no remarkable perioperative complications.

Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase.

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.

Serum and bile bilirubin pigments in the differential diagnosis of Crigler-Najjar disease.

OBJECTIVE: To differentiate between Crigler-Najjar (CN) disease types 1 and 2. DESIGN: The patterns of serum bilirubins, bile pigment composition, and phenobarbital response were studied. PATIENTS: Three infants, affected by high serum unconjugated bilirubin concentrations, previously classified as type 1 CN. METHODS: Serum and bile bilirubin pigment composition, both before and after phenobarbital (PB) treatment, were determined by alkaline methanolysis and high-pressure liquid chromatography. PB was given for at least 3 weeks by oral administration (5 mg/kg bw per day). RESULTS: No diconjugated bilirubin was found either before or after PB treatment in the serum of the three studied infants. In two patients traces of monoconjugated bilirubin were detected before PB therapy, and the ratio of conjugated/total bilirubin (percent) was increased by the PB response. In the third patient, traces of monoconjugated bilirubin appeared only after PB administration. However, the serum unconjugated bilirubin concentration decreased significantly only in the second patient, following the second cycle of PB treatment, leading to the diagnosis of type 2 CN. The analysis of the methyl ester derivatives of bile pigments was also performed on bile samples obtained in two patients by Entero-Test (R) both before and after PB treatment. An absolute increment in monoesterified bilirubin concentration was found after PB administration, although the percent concentration increased in one case and decreased in the other. No diesterified bilirubin was detected in the bile samples. CONCLUSIONS: The present results show that in types 1 and 2 CN disease it is possible to detect traces of monoconjugated but not diconjugated bilirubin both in serum and in bile. Whereas PB treatment is effective in slightly increasing the serum monoconjugated bilirubin concentration even in type 1 CN disease, the diagnosis of type 1 or 2 is based on finding a substantial decrease of serum unconjugated bilirubin following PB administration.

A new type of defect in the gene for bilirubin uridine 5'-diphosphate-glucuronosyltransferase in a patient with Crigler-Najjar syndrome type I.

Crigler-Najjar syndrome (CN) type I, which is characterized by the complete absence of bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT) activity, is inherited as an autosomal recessive trait associated with unconjugated hyperbilirubinemia. Phenobarbital has no effect on the bilirubin concentration in the serum of patients with CN type I. Recently, cDNA for two human liver bilirubin UGT (UGT1A and UGT1D) were isolated, and their genetic organization was determined. The UGT1A (UGT1*1) and UGT1D (UGT1*4) genes each have a unique exon 1, whereas exons 2-5 are common to both genes. It has been predicted that some defect in the exons common to both genes is responsible for the absence of UGT1A and UGT1D activities in CN type I, and five cases with such a mutation have been reported. We describe here a new type of defect in the gene for bilirubin UGT in a patient with CN type I, namely, an abnormality in the exon 1 that is characteristic of the UGT1A gene. This mutation is a single nucleotide substitution, that is, C is changed to A at base position 840 in UGT1A cDNA, and this change results in a stop codon. Our patient is homozygous for the defect, and his nonconsanguineous parents and elder brother, who are clinically normal, are heterozygous for the defective allele. No mutation was detected in any exons of the UGT1D gene. Our results suggest that a homozygous nonsense or deletion mutation is detected not only in the exons common to UGT1A and UGT1D genes but also in unique exon 1 of UGT1A in CN type I.

An unusual case of Crigler-Najjar disease in the adult. Classification into types I and II revisited.

We present the case of a 25-year-old man with Crigler-Najjar disease who had since birth a marked unconjugated hyperbilirubinemia without bilirubin overproduction, without any neurological involvement and in whom phenobarbital administration failed to produce any effect. Analysis of his biliary bile pigments on two occasions showed (i) a decrease excretion of bilirubin, as indirectly suggested by a high ratio of biliary bile acids over total bilirubin; (ii) an increase in unconjugated bilirubin IX alpha quantitated by thin-layer chromatography (TLC) following alkaline methanolysis and by direct extraction and TLC of the tetrapyrroles; (iii) a high proportion of bilirubin monoconjugates whereas the excretion of diconjugates was very low. Classification of the present patient into Crigler-Najjar disease type I or II was not possible. The most striking and practical difference among the various cases of Crigler-Najjar disease remains the response to phenobarbital. Among cases of Crigler-Najjar disease which respond to enzyme induction and Gilbert's syndrome, the continuous spectrum suggests a common defect.